Biolaminin 521 LN

Biolaminin 521 LN (LN521)

Human recombinant laminin 521


Recreating the natural cell niche is key to successful cultivation. The physical, topological, and biochemical expression of the different laminin isoforms in the BM is heterogeneous and tissue specific. 

Biolaminin 521 LN (LN521) is the natural laminin for pluripotent stem cells and therefore reliably facilitates self-renewal of human ES and iPS cells in a chemically defined, feeder-free and animal component-free stem cell culture system. LN521 enables efficient single-cell passaging of genetically stable and pluripotent stem cells without need of any apoptosis inhibitors for superior quality of your cells and studies. 

After birth, α5-laminins represent the most common laminins in the body and LN521 supports many diverse tissue cell types, such as cells from pancreas, vascular, nervous and muscular systems.

BIOLAMININ 521 PRODUCT SHEET

 

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Amount
Size
Price
Oty
100 µg 1 x 1 ml €55
500 ug 1 x 5 ml €210
2,5 mg 5 x 5 ml €985
17,5 mg 35 x 5 ml €5850
For larger quantities contact us

Estimated shipping time
1-5 working days

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SIZE GUIDE

Not sure how much laminin you need? To make it easy, we have created a tool where you can calculate the amount needed for your experiments. Just choose culture well format and fill in the desired coating concentration to see the amount required.

FORMAT
CONC. (ug/cm2).
NO OF WELLS
AMOUNT (uq)

Features and Specifications:

  • Research grade substrate

  • Defined and animal component–free to the primary level

  • Biologically relevant hPSC culture environment

  • Homogenous and genetically stable hPSC cultures

  • Positive effect on stabilizing and homogenizing pluripotent gene expression profiles between hES cell lines
  • Easy and flexible culture system

  • Consistent and reliable performance

  • Rapid scale-up

  • Supports a weekend-free feeding regime

  • Efficient differentiation and enhanced cell maturation, polarization and organization

  • Scientifically proven

 

LN521 cell culture application note

 

A BIOLOGICALLY RELEVANT CULTURE ENVIRONMENT

Biolaminin 521 LN is a full-length, human, recombinant laminin 521 substrate, the only one of its kind on the market, providing an optimal environment for feeder-free culture of hPSCs. Laminin 521 is a key cell adhesion protein of the natural stem cell niche, expressed and secreted by hPSCs in the inner cell mass of the embryo. The Biolaminin 521 substrate recreates a biologically relevant hPSC milieu in vitro , promoting attachment, high survival, and robust long-term self-renewal of hPSCs. In addition, the more authentic culture environment primes the pluripotent stem cells for more efficient differentiation, enhanced cell maturation, polarization and organization of specialized cell types. The laminin 521 cell culture substrates have a positive effect on stabilizing and homogenizing pluripotent gene expression profiles between hES cell lines, providing a more controllable and robust culture system compared to feeder and many other feeder-free culture systems. Via the interaction with relevant cell surface receptors, laminins activate cell signalling cascades (e.g. PI3K/Akt pathway), giving consistent cellular responses and improved cell functionality, ultimately saving researchers both time and cost.

The LN521 stem cell substrate enables high expansion rate, monolayer homogenous growth, and maintained pluripotency. Cells are genetically stable without karyotypic abnormalities. hESC and iPSC grow faster on LN521 compared to cells cultured on feeders or on another feeder-free matrice, facilitating automation. Human pluripotent stem cells can routinely be cultured as single cells without the addition of ROCKi. LN521 also supports efficient clonal survival and expansion of hES and iPS cells, greatly facilitating the study of genomic alterations and manipulations in pluripotent cells. Furthermore, renders more efficient reprogramming of specific tissue cells to iPSCs.


The Biolaminin 521 substrate is more flexible, easier to use and less laborious compared to feeder-based culture systems and is compliant with any commercially available serum-free medium. The cells grow in a homogeneous monolayer and are easy to monitor, with maintained pluripotency and genetic integrity. The robust and flexible culture environment allows an operator-independent culture maintenance and reliable, standardized protocols, which can easily be adapted to automation platforms. The robust support of pluripotent stem cells by LN521 also enables maintenance of hPSCs withour weekend feeding. Read more.


After birth, laminin 521 represent one of the most common laminins in the body and together with the other laminin isoforms, it supports efficient expansion and differentiation of many diverse tissue cell types.

 


Read more about different laminin cell culture applications


Download cell culture applications poster

biorelevant stem cell culture

Human stem cell culture

Easy culturing of human ES and iPS cells in a 
biorelevant and animal component-free environment 

effective clonal culture

Clonal cell culture

Efficient clonal culture and derivation of
human pluripotent stem cells 

Biological relevance


 natural substrate for stem cell culture

Laminin 521 is a key cell adhesion protein of the natural stem cell niche, expressed and secreted by hPSCs in the inner cell mass of the embryo. LN521 recreates the biologically relevant milieu in vitro and therefore supports robust expansion of human ES and iPS cells. 

  

 


 

Easy and flexible protocols

easy and flexible cell culture protocols

The single-cell split is easy, fast and forgiving, making anyone suitable to culture hPSCs. When using LN521, your cell culture protocol can easily be made totally defined and xeno-free with your choice of culture medium and dissociation reagent. For reduced labor and cost, the LN521 matrix supports weekend-free feeding.

  1. Coat plates with LN521.

  2. Wash the cells with PBS and add dissociation reagent of choice - incubate

  3. Centrifuge the single-cell suspension and resuspend the pellet in fresh medium of choice

  4. Seed as single-cell suspension on fresh LN521 coated plates


 

Cost-effective stem cell culturing

 Laminin-521 cost effective stem cell culture

The specific LN521 binding of the α6β1 integrin enables high cell survival and self-renewal and hPSCs grow faster on LN521 compared to other feeder-free matrices. Due to faster growth rate and higher cell yield, the total cost (medium and matrix) per cell for an average passage is lowest for LN521. Read more about cost-effective stem cell culture

Long-term efficient expansion

Rapid and robust stem cell expansion

hESC and iPSC grow faster on LN521 compared to other feeder-free matrices, facilitating automation. The specific LN521 binding of the α6β1 integrin enables high cell survival and self-renewal. Cells expand quickly and reach 100% confluency after only 4 days after a 1:10 split. You will thus have 10.000 fold more cells after only 4 passages, making the cell quantities of low-passage hPSCs needed for clinical applications possible.


 

Genetic stability 

genetically stable stem cell culture
 

With LN521 hPSCs can be seeded as single cells at low densities and cultured long-term without introduction of genetic abnormalities. Cells have been kept on LN521 for more than 130 passages with stable karyotypes. Importantly, when LN521 is used for the derivation of new hESC lines, the embryo is not destroyed and the lines originating from a single blastomer are genetically stable.

 

 

 

 

 


 

Homogenous hPSC monolayer 

homogenous stem cell culture without spontaneous differentiation

PSCs plated as single-cell suspension on LN521 grow as a homogenous monolayer without any abnormal genetic aberrations. The specific LN521 binding of the α6β1 integrin enables high cell survival and self-renewal and once adapted to the LN521 matrix, hPSCs can routinely be cultured as single cells without the addition of ROCKi. LN521 also support clonal survival and is a good substrate for derivation and gene editing.  


 

Homogenous and stable pluripotent gene expression 


 

stem cell culture without spontaneous differentiation 

 

Culture on LN521 significantly reduced the variation of pluripotency marker expression compared to culture on hFFs (fibroblasts). Moreover, hES cells cultured on LN521 are more homogenous, attached better and grew faster compared to hES cells cultured on other substrates. hPSCs can be cultured to near confluence on LN521 without spontaneous differentiation and there is no need to remove differentiated cell areas. LN521 have a homogenizing and stabilizing effect on pluripotent gene expression profiles between hES cell lines and provide the first step towards generating more controllable, robust and clinically safe culture conditions.




 

Read the full-length Nature Communications article on biorelevant and effective stem cell culture

COATING PLATES

See instructions for coating protocol, recommended coating volumes and concentrations.


hPSC CULTURE ON LN521

See instructions for stem cell culture on laminin 521, including:
- transfer
- passaging
- thawing
- cryopreservation of hPSCs on LN521. 


Embryoid body formation

Protocol for embryoid body formation from hPSCs cultured on LN521

 

 

IMPORTANT NOTES

  • "For Research Use Only"

  • All procedures should be done under sterile conditions using aseptic techniques

  • Avoid long exposure of the protein to ambient temperatures

  • The laminin stock solution is stable for 3 years when stored at -20°C to -80°C. Thawed, undiluted laminin stock is stable for at least 3 months when stored at +2°C to +8°C under aseptic conditions

  • Repeated freeze thawing should be avoided. If desired, the laminin stock can be dispensed into working aliquots and stored at -20°C to -80°C. 

  • For your convenience, the coated plates can be kept for up to 4 weeks when stored aseptically at +2°C to +8°C

  • The protocols can easily be made totally defined and xeno-free with your choice of culture medium and dissociation reagent

  • It is important that the cells transferred to the LN521 matrix are of high quality

  • Some hPSC lines transferred to the LN521 matrix, might require an adaptation period before they can be cultured according to the single-cell passaging protocol

  • Once adapted to the LN521 matrix, hPSCs can routinely be cultured as single cells without ROCKi

  • The LN521 matrix facilitates long-term self-renewal of hPSC without weekend feeding. For reduced labor and cost, follow the reduced feed protocol.

Troubleshooting

Laminin plate coating

An uneven cell spread, it’s often a coating issue and could be caused by the following:

  • To low coating concentration used. Increase the coating concentration is high enough to support an even cell growth.
  • DPBS Ca--/Mg-- has been used. We recommend to use DPBS with Ca2+ and Mg2+ since divalent cations are important for the protein structure and function. 
  • NUNC-plates was used. Most plastic work well for laminin coating but we know the laminin coating is not compatible with some NUNC-plates. SARSDET and Corning plate usually work well.
  • Bad coating coverage/the plate has dried out. Ensure that the entire surface is covered by the laminin coating solution when preparing fresh plates. Also, do not let the plate dry out as this will inactivate the laminin coating. Too long time in the incubator or long storage without sealing could cause too much evaporation so that part of the plate dries out (often in the center).

 
Transfer from other substrates

  • We recommend to transfer the cells as single cells (or as small clumps) and always with the addition of ROCKi for the first few (3-5) passages. Once the cells are adapted to the LN521 matrix, the cells can be cultured as single cells without ROCKi. This may take up to 5 passages.
  • If the cells are hard to adapt, try increasing the coating concentration to 10 ug/ml and seed at a higher cell density 50,000 – 100,1000 cells/cm2. Once the cells are adapted a lower coating and seeding concentration often can be used.
  • It is important that the cells transferred to the LN521 matrix are of high quality. LN521 will generally also support differentiated cells so carefully select only undifferentiated cell areas for transfer. 
  • LN521 work well in combination with most commercial media brands. However, it is to be expected that cell morphology will look different dependent on the medium used for culture. 


hPSC splitting and seeding

  • We recommend single-cell passage or passage as small clumps. 
  • Stem cells are sensitive and when using enzyme, do not treat the cells too long as that will damage the cells. Cells attach tighter on laminin compared to other matrices and scraping or pipetting without first loose up cells can affect cell integrity and viability which could result in less attachment next day. Less confluent cells need shorter treatment time whereas more confluent cells might need longer treatment time. The cells should detach easily without too much pipetting needed. Do not use too much mechanical force (extensive pipetting or scraping) as that will damage the cells. Increase the dissociation reagent incubation time rather than increasing the force.  If the cells stick very hard to the LN521 surface, try to lower the coating concentration.
  • After seeding: most cells should have attached after 1 hour and the cells should be evenly distributed over the entire plate. If there is a lot of cell death after seeding, the cells have most likely been treated too harsh during splitting.
  • The day after seeding: the cell has migrated and should have formed small colonies and will continue to expand as a homogenous monolayer. Cells cultured on the LN521 matrix are ready to be passaged when cell culture is 60-99% confluent. Depending on the cell line, seeding density and on the medium used, cultures are usually passaged 3-6 days after seeding.

Product name

Biolaminin 521 LN

 

Product code

LN521

 


Declaration

For research use only

 


Documents

MSDS

CoA

 

Storage

-20°C to -80°C

 

Stock concentration

0.1mg/ml

 


Appearance

Clear, colourless, buffered solution with a 
pH of 7.2 with 10% glycerol and 0.02% NaN3

 

Shipping Condition

Dry Ice

Protein name

Laminin 521 (Laminin-11)

 

Classification

Defined and animal component-free, human 
recombinant protein 

 

Product application

Culture of human embryonic and induced pluripotent stem cells (hESC and hiPSC), mesenchymal stem cells (MSC) and most anchorage-dependent progenitor cell types. Differentiation and maintenance of specialized cells, such as hepatocytes, cardiomyocytes and neurons.

Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment

Rodin S., Antonsson L., Niaudet C., Simonson O.E., Salmela E., Hansson E.M., Domogatskaya A., Xiao Z., Damdimopoulou P., Sheikhi M., Inzunza J., Nilsson A.S., Baker D., Kuiper R., Sun Y., Blennow E., Nordenskjöld M., Grinnemo K.H., Kere J., Betsholtz C., Hovatta O., Tryggvason K. 
Nature Communications, 2014


Monolayer culturing and cloning of human pluripotent stem cells on laminin-521 based matrices under xeno-free and chemically defined conditions

Rodin S., Antonsson L., Hovatta O., Tryggvason K. 
Nature Protocols, 2014


a-5 Laminin Synthesized by Human Pluripotent Stem Cells Promotes Self-Renewal

Laperle A., Hsiao C., Lampe M., Mortier J., Saha K., Palecek S.P., and Masters K.S. 
Stem Cell Reports, 2015

Laminin 521 stabilizes the pluripotency expression pattern of human embryonic stem cells initially derived on feeder cells

Albalushi H., Kurek M., Karlsson L., Landreh L., Rós Kjartansdóttir K., Söder O., Hovatta O., Stukenborg J-B. 
Stem Cell International, 2017


Niche-derived laminin-511 promotes midbrain dopaminergic neuron survival and differentiation through YAP

Zhang D., Yang S., Toledo E.M., Gyllborg D., Saltó C., Villaescusa J.C., Arenas E. 
Sci Signal. 2017

 

A defined xeno-free and feeder-free culture system for the derivation, expansion and direct differentiation of transgene-free patient-specific induced pluripotent stem cells.

Hong Fang Lu, Chou Chai, Tze Chiun Lim, Meng Fatt Leong, Jia Kai Lim, Shujun Gao, Kah Leong Lim, Andrew C.A. Wan.
Biomaterials, 2014

 


Optimization of slow cooling cryopreservation for human pluripotent stem cells.

Takamichi Miyazaki, Norio Nakatsuji and Hirofumi Suemori.
Genesis, 2013

 

 

Xeno-free culture of human pluripotent stem cells.

Bergström R, Ström S, Holm F, Feki A, Hovatta O.
Methods Mol Biol, 2011